Project description:A laboratory colony of Phlebotomus perniciosus sand flies was maintained. Sand flies were infected with cultured Leishmania infantum promastigotes in stationary phase. Ten infected sand flies were dissected after 5 days and promastigotes within the gut pooled. The cells were immediately washed in PBS once and lysed in TRIzol reagent (Life Technologies). RNA isolation was completed according to the manufacturer's instructions, obtaining 63ng. RNA-seq libraries were generated using the spliced leader sequence for second strand synthesis (Cuypers et al., 2017; Haydock et al., 2015), thus allowing for specific amplification of sequences from L. infantum promastigotes, thus avoiding contamination with material from the sand fly gut. Single-end sequencing was performed in an Illumina HiSeq2500 instrument and data analysis was conducted using bowtie2, samtools, featureCounts and Geneious. The main findings are: i) substantial differences in differential gene expression between sand fly-derived (sfPro) and cultured (acPro) promastigotes; and ii) over-expression of genes involved in metacyclogenesis in sfPro vs. acPro, including gp63 genes, autophagy genes, etc.
Project description:Leishmania infantum (Kinetoplastida:Trypanosomatidae) is the etiological agent of zoonotic visceral leishmaniasis in the Mediterranean basin. The motile promastigote stage infects the hematophagous sand fly vector host and amastigotes survives and multiplies within phagocytes of the mammalian host. Promastigotes are routinely cultured in liquid undefined media and are considered to mimic the environment within the sand fly gut. We have put this to the test by high-throughput gene expression profiling by shotgun DNA microarrays generated in our laboratory. This has been possible thanks to RNA amplification.
Project description:The leishmaniases are globally important parasitic diseases for which no human vaccines are currently available. To facilitate vaccine development, we conducted an open label observational study to establish a controlled human infection model of sand fly-transmitted cutaneous leishmaniasis caused by L. major. Between 24th January and 12th August 2022, we exposed 14 (8F, 6M) participants to infected sand flies. The primary objective was to demonstrate effectiveness (attack rate) and safety (absence of CL lesion at 12 months), whereas secondary and exploratory objectives included rate of lesion development, parasite load and analysis of local immune responses using immunohistology and spatial transcriptomics. We estimated a take rate for CL development of 64% (9/14) based on all participants, increasing to 82% (9/11) if only participants with confirmed bites are included. Lesion development was terminated by therapeutic biopsy in 10 participants with confirmed bites. 30% (3/10) had either one (2/10) or two (1/10) lesion recurrences between 4-8 months after biopsy that were treated successfully with cryotherapy. No severe or serious adverse events were recorded, but scarring was evident as expected. All participants were lesion-free at long-term (>12 month) follow up. We provide the first comprehensive map of immune cell distribution and cytokine/chemokine expression in human CL lesions, revealing discrete immune niches. This controlled human infection model offers opportunities for rapid vaccine candidate selection and a greater understanding of immune-mediated protection and pathology.
Project description:Arbovirus transmission by sand flies is a growing public health concern, yet the early skin events shaping infection outcomes remain undefined. We establish a new mouse model of Toscana virus (TOSV) infection that incorporates sand fly salivary factors to mimic natural transmission. Saliva from two distinct sand fly genera significantly enhanced infection and promoted neurological signs and joint inflammation, recapitulating key features of human TOSV disease. In the skin, dermal macrophages and fibroblasts were the main infected cell types, but only fibroblasts generated infectious virus. Saliva reprogrammed fibroblasts into a wound-healing state permissive to viral replication, driving local viral amplification, systemic spread, and thereby clinical disease. These findings identify skin fibroblasts as central determinants of host susceptibility and reveal that sand fly saliva actively remodels the skin to exacerbate viral pathogenesis. This work redefines the skin’s role in sand fly-transmitted infection and highlights new targets for therapeutic and vaccine development.